Proteins are linear polymers of amino acids. Since the polymerization reaction which produces a protein results in the loss of one molecule of water from each amino acid, proteins are often said to be composed of amino acid "residues." Natural protein molecules may contain as many as 20 different types of amino acid residues, each of which contains a distinctive side chain. The sequence of amino acids in a protein defines the primary structure of the protein.
Proteins fold into a three-dimensional structure. The folding is determined by the sequence of amino acids and by the protein's environment. The remarkable properties of proteins depend directly from the protein's three-dimensional conformation. Thus, this conformation determines the activity or stability of enzymes, the capacity and specificity of binding proteins, and the structural attributes of receptor molecules. Because the three-dimensional structure of a protein molecule is so significant, it has long been recognized that a means for stabilizing a protein's three-dimensional structure would be highly desirable.
The three-dimensional structure of a protein may be determined in a number of ways. Perhaps the best known way of determining protein structure involves the use of the technique of X-ray crystallography. An excellent general review of this technique can be found in Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, N.J. (1971) pp221-239) which reference is herein incorporated by reference. Using this technique, it is possible to elucidate three-dimensional structure with remarkable precision. It is also possible to probe the three-dimensional structure of a protein using circular dichroism, light scattering, or by measuring the absorption and emission of radiant energy (Van Holde, Physical Biochemistry, Prentice-Hall, N.J. (1971)). Additionally, protein structure may be determined through the use of the techniques of neutron defraction, or by nuclear magnetic resonance (Physical Chemistry, 4th Ed. Moore, W. J., Prentice-Hall, N.J. (1972) which reference is hereby incorporated by reference).
The examination of the three-dimensional structure of numerous natural proteins has revealed a number of recurring patterns. Alpha helices, parallel beta sheets, and anti-parallel beta sheets are the most common patterns observed. An excellent description of such protein patterns is provided by Dickerson, R. E., et al. In: The Structure and Action of Proteins, W. A. Benjamin, Inc., Calif. (1969). The assignment of each amino acid to one of these patterns defines the secondary structure of the protein. The helices, sheets and turns of a protein's secondary structure pack together to produce the three-dimensional structure of the protein. The three-dimensional structure of many proteins may be characterized as having internal surfaces (directed away from the aqueous environment in which the protein is normally found) and external surfaces (which are in close proximity to the aqueous environment). Through the study of many natural proteins, researchers have discovered that hydrophobic residues (such as tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, or methionine) are most frequently found on the internal surface of protein molecules. In contrast, hydrophilic residues (such as asparate, asparagine, glutamate, glutamine, lysine, arginine, histidine, serine, threonine, glycine, and proline) are most frequently found on the external protein surface. The amino acids alanine, glycine, serine and threonine are encountered with equal frequency on both the internal and external protein surfaces.
Proteins exist in a dynamic equilibrium between a folded, ordered state and an unfolded, disordered state. This equilibrium in part reflects the short range interactions between the different segments of the polypeptide chain which tend to stabilize the protein's structure, and, on the other hand, those thermodynamic forces which tend to promote the randomization of the molecule.
Metal ions have long been known to stabilize proteins by binding at specific sites in the tertiary structure. Indeed, many proteins isolated from thermophilic organisms have been shown to contain calcium ion binding sites. These sites are responsible for the enhanced stability that these proteins require in order to function in the elevated temperature milieu in which they are found. For example, thermolysin, a neutral protease from the thermophilic organism Bacillus thermoproteolytichs, was found to contain four calcium binding sites. By studying the calcium ion dependence for the rate of thermal inactivation, it has been possible to estimate the collective contribution of these sites to the G of unfolding to be between 8.1 and 9.2 Kcal/mole (Vorrdouw et al., Biochemistry 15:3716-3724 (1976)).
For example, Serpersu et al., Biochemistry 26:1289-1300 (1987) replaced the carboxylate ligand of residue aspartic acid 40 in the calcium binding site of staphylococcal nuclease with a glycine resulting in a 7.4 fold decrease in affinity for calcium ions. Similarly, Tsuju et al., Proc. Nat'l Acad. Sci. USA, 83:8107-8111 (1986) changed glycines in the vicinity of the calcium binding loops in the photoprotein aequorin to positively charged arginines to cause a decrease in the affinity of metal binding at these sites. It would, however, be desirable to design proteins with increased stability by increasing the binding affinity of divalent metal cations at a specific site in the protein through specific amino acid substitutions.